Varga Andrea, Szabó Judit, Flachner Beáta, Roy Béatrice, Konarev Peter, Svergun Dmitri, Závodszky Péter, Périgaud Christian, Barman Tom, Lionne Corinne, Vas Mária
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina Str. 29, P.O. Box 7, H-1518 Budapest, Hungary.
Biochem Biophys Res Commun. 2008 Feb 22;366(4):994-1000. doi: 10.1016/j.bbrc.2007.12.061. Epub 2007 Dec 26.
l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k(cat) and K(m) values of 685s(-1) and 0.27mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg(2+)), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements.
L-核苷类似物是天然D-核苷的镜像，是一类新型的抗病毒和抗癌药物。在细胞中，它们必须磷酸化成为具有药理活性的三磷酸形式，最后一步似乎涉及人3-磷酸甘油酸激酶（hPGK）。在此，我们展示了模型化合物L-MgADP与hPGK相互作用的稳态动力学和生物物理研究。L-MgADP是一种良好的底物，其k(cat)和K(m)值分别为685s(-1)和0.27mM。双重抑制研究表明，L-MgADP结合到特定的腺苷结合位点，并保护hPGK分子的构象免受热变性影响，这通过微量热法检测到。d-和l-对映体在酶活性位点相互作用的结构细节有所不同（例如Mg(2+)的影响），但这些差异并不妨碍催化循环的发生，如小角X射线散射测量所示，催化循环伴随着铰链弯曲结构域的闭合。
